Production of most crop species is limited by the ravages of pests and diseases. Considerable expense is involved in the protection of crops from these organisms and many current conventional breeding programs are directed at increasing host plant resistance to a variety of invertebrate pests and fungal or viral diseases. Traditional sources of host plant resistance are limited to the same species or species closely related to the crop, but with the advent of genetic engineering novel sources of resistance outside the crop genera are being accessed. Two classic examples are the insecticidal protein genes from Bacillus thuringiensis active against a multitude of insect species (Perlak et al., 1990) and the viral coat protein genes that confer tolerance to a variety of related viral diseases (Powell et al., 1986). Transgenic plants expressing these genes have enormous potential markets once the regulatory hurdles are overcome for their large scale release into agriculture.
New sources of host plant resistance are being sought from a variety of sources, and work leading to the present invention has led to the isolation and characterisation of a glucose oxidase gene from the fungus Talaromyces flavus that has potential for the control of both fungal diseases and arthropod pests when expressed in transgenic crop plants. The particular examples investigated to date are for the control of the cotton pathogen, Verticillium dahliae, the causal agent of verticillium wilt disease, and for the control of Helicoverpa spp. which are economically important Lepidopteran pests of cotton and most summer crops. Use of the glucose oxidase gene for the control of other fungal diseases or pests such as nematodes, mites, aphids, whiteflies, jassids or mirids which are susceptible to hydrogen peroxide produced by glucose oxidase activity is also possible.
Verticillium Wilt as a Cotton Pathogen
Verticillium wilt is a wide-spread disease which affects many different plant species. It is caused by the soil fungus Verticillium dahliae (Leb.), an imperfect fungus first isolated from diseased dahlias in 1913 (Muller, 1928). Isolates of the species vary widely in both morphology and pathogenicity but all produce small, hard black structures called microsclerotia. These structures are composed of melanized cells which store many nutrients and are the means by which V. dahliae survives in the soil.
V. dahliae does not grow saprophytically through the soil, but conidia and microsclerotia germinate in soil if root exudate from desirable plant species are present. The mycelium then invades the plant, entering through the cap of the root region of elongation, root hairs or lower hypercotyl region. In all cases both intercellular and intracellular invasion occurs. In "susceptible" hosts, the fungus successfully penetrates the vascular system of the plant. Here hyphal multiplication occurs and conidiospores are generated which then travel upwards through the xylem thus spreading the fungus rapidly through the vascular system of the plant.
Once spread throughout the vascular system the main effect of the pathogen is to disrupt the passage of water through the stem. This may be attributed to the physical presence of the mycelium of the pathogen, the development of tyloses that block the xylem or to gummosis within the vessels. Further xylem occlusion is caused by high molecular weight polysaccharides produced by the pathogen or cleaved from the plant walls by hydrolytic enzymes. Together this can result in a 40 to 60-fold increase in the resistance of the stem to water flow; thus the characteristic wilting of leaves occurs (Ayer and Racok, 1990).
In cotton more obvious symptoms include a yellowing of lower leaves, vascular discolouration and stunting of plant growth. Some V. dahliae isolates also cause severe defoliation of the cotton plant. On the basis of heterokaryon incompatibility tests these isolates fall into a separate group from the less severe, non-defoliating isolates (Puhalla, 1979). All Australian isolates so far examined belong to the less severe, non-defoliating group, however they are still capable of causing a significant reduction in lint yield.
Controlling verticillium wilt in cotton in Australia has frequently been a problem, particularly in the cooler growing regions (New South Wales and Southern Queensland) where cooler, wetter seasons promote more vigorous fungal growth. The microsclerotia produced are resistant to many soil fumigants and remain viable in the soil for many years. Crop rotation is also not a satisfactory control measure because of the wide host range of the fungus. No natural resistance to Verticillium has been identified in cotton, any natural tolerance that has been found is being exploited by the breeders.
Biological control of the pathogen with other microbes is a possible solution. Marios et al. (1982) investigated the potential of over 30 different soil fungi to control verticillium wilt development in eggplant under field conditions. An isolate of Talaromyces flavus was identified that reduced disease symptoms by approximately 70% in two separate field situations. Trials have shown that T. flavus is able to occupy the rhizosphere of Australian cotton. Further trials will need to be done to determine if this naturally occurring strain or improved engineered strains can control verticillium wilt. However, even if successful, T. flavus itself may not be a suitable control agent as large scale seeding of soil in which cotton is to be grown may be impractical and other microbes that live in close association with the cotton plant may be more effective delivery systems.
Helicoverpa spp. are serious pests of cotton and other summer crops.
Helicoverpa armigera and H. puctigera are two of the most economically important insect pests in many cropping systems in Australia (Fitt, 1989). Their larvae cause considerable damage in crops such as cotton and their control requires the application of vast amounts of chemical pesticides. Many of these pesticides are becoming ineffective due to the development of resistance by the insects and sources of host plant resistance in the crop would be extremely valuable, both economically and environmentally.
Adult moths lay their eggs singly, generally on the young growing tips of the plant. When the neonates emerge they feed on the egg case and then migrate to the young flower buds (squares) and begin to graze. A single larvae may visit several flower buds rather than staying on one square for extended periods of time. Relatively small amounts of feeding damage cause flower abortion and hence the economic threshold for larvae on plants is relatively low. As little as one larvae per meter of cotton row can have a significant effect on yield, so relatively low infestations must be sprayed to control the insect. As indicated below, glucose oxidase is toxic to these caterpillars when incorporated into synthetic diets containing a source of glucose and so may be a useful gene to express in young cotton tissues such as leaves and squares.
Talaromyces flavus is a glucose oxidase secreting ascomycete.
Talaromyces flavus (Klocker) Stolk and Samson (anamorph Penicillium dangeardii Pitt, usually reported as P. vermiculatum Dangeard) is the most common species of its genus. This ascomycete is frequently isolated from soil, although it may also occur in other organic substrates. It is widely distributed around the world but is more commonly found in warmer regions. It has been reported as a potential biocontrol agent for several other fungal pathogens, Rhizoctonia solani (Boosalis, 1956) and Sclerotinia sclerotiorum (McLaren et al., 1986).
In the above cases, T. flavus controls the pathogens by mycoparasitism, that is T. flavus parasitises its fungal host for nutrient gain. Infection studies of S. sclerotiorum and R. solani have shown T. flavus coils around the host developing hyphal branches which then penetrate the host's cells. Deterioration of the cytoplasm follows with the infected cells eventually collapsing, although the cell walls remain intact. Transmission electron micrographs of V. dahliae microsclerotia parasitised by T. flavus have similarly shown cell invasion and lysis taking place only at the contact sites between the host's cells and T. flavus hyphal tips (Madi et al., 1989).
The mechanisms involved in the parasitic interactions are unclear. However Fravel et al., (1987) found T. flavus secreted a metabolite into liquid medium which in the presence of glucose was toxic to microsclerotia and inhibited radial growth of Verticillium mycelia. The active component was subsequently identified as glucose oxidase secreted from fungal hyphae (Kim et al., 1988). This enzyme has now been shown to inhibit other fungi including several of the Pythium species, Rhyzoctonia solani and Sclerotinia minor (Kim et al., 1990.sup.a,b).
Glucose oxidase leads to the production of hydrogen peroxide, (H.sub.2 O.sub.2) as a by-product of glucose oxidation: ##EQU1##
When added to growth media, hydrogen peroxide inhibited microsclerotial germination and mycelial growth. The other reaction components, glucose oxidase (no glucose present), gluconic acid and glucose did not cause inhibition (Kim et al., 1988). Thus the antifungal activity of glucose oxidase is due to the hydrogen peroxide it produces. However when the peroxide scavenger catalase was added to the culture filtrate of T. flavus there was only a loss of 50% of its toxic activity towards V. dahliae (Madi et al., 1989). Thus T. flavus may produce other agents toxic to V. dahliae. T. flavus has been found to excrete a range of lytic enzymes including cellulases, .beta.-1-3-glucanases and chitinase. Therefore the antagonistic activity of T. flavus towards V. dahliae may be due to a combined effect of lytic enzymes and toxic metabolites. The glucose oxidase may act by inhibiting the Verticillium and thus predispose the hyphae to infection before contact occurs.
Glucose oxidase the active agent in the antagonism by T. flavus of V. dahliae.
The enzyme glucose oxidase is known to be produced by different species of Aspergillus and Penicillium, by Talaromyces flavus and by the basidiomycete Phanerochaete chryosporium, (white rot fungus). In P. chryosporium (found in wood), the hydrogen peroxide produced is required by a ligninase enzyme for the degradation of lignin. In the other fungi little is known about the enzyme's biological function. They may produce enzymes which utilise hydrogen peroxide as P. chryosporium does. In this case the enzyme's ability to inhibit various other soil fungi may be a secondary effect, however it would benefit the host in certain competition situations.
Glucose oxidase has been purified from each of the four fungal genera known to produce it. In all cases, the enzyme is a dimeric flavoprotein with an optimum pH of 5.0. The most distinct enzyme is that of P. chryosporium. Unlike the others it is not glycosylated and although glucose is its primary substrate it is also induced to a smaller degree by sorbose, xylose and maltose (33, 13 and 7% respectively) (Kelly and Reddy, 1986). The other enzymes are highly specific for .beta.-D-Glucose.
Glucose oxidase from T. flavus has a relative molecular weight of 164,000 (subunit molecular weight 71,000) (Kim et al., 1990). This is similar to that of Penicillium amakienase (150,000) and Aspergillus niger (152,000), (Nakamura and Fujiki, 1968). It is stable from pH 3.0 to 7.0, unlike A. niger which is restricted to pH 4.5 to 6.5. Six isozymes with pl values of 4.40 to 4.55 have been detected. These are thought to be due to differences in sugar residues as opposed to differences in amino acid sequence. It has a relatively low affinity for glucose with a Km for .beta.-D-glucose of 10.9 mM. This is however a higher affinity than that of A. niger which has a Km for .beta.-D-glucose of 27 mM.
The gene for glucose oxidase from A. niger has been cloned by several groups (Kriechbaum et al., 1989, Frederick et al., 1990, Whittington et al., 1990). The structural gene consists of 1815 bp encoding 605 amino acid residues. The mature protein contains 583 amino acids, the difference being due to 22 amino acids which comprise the secretion signal presequence. No introns were present in the coding region. The gene has been introduced into Aspergillus nidulans and the yeast Saccharomyces cerevisiae where it provided the novel capacity to produce glucose oxidase. It has also been reintroduced into A. niger where increased copy number increased glucose oxidase production.
In the past, biological control of pests and diseases has focussed on natural biocontrol agents such as antagonistic bacteria and fungi or viruses. It has now been found that the effectiveness of these agents can be enhanced if they are engineered to express the glucose oxidase activity. The present invention therefore includes the use of other vectors for delivering the glucose oxidase activity to the pest or pathogen, such as root or leaf colonising micro-organisms which could be beneficial bacteria or fungi that live around the plant and that could exert their effects on plant pests in the rhizosphere or phylloplane or, for example, insect specific viruses that could be sprayed onto the plants.